Actin filaments are essential structural elements in cells and actin filament assembly produces forces for some types of cellular movements such as locomotion and endocytosis. Cellular movements are essential for cell division, embryonic development and defense against microorganisms. Movement of cells out of primary tumors is the chief cause of mortality in cancer. This grant supports basic research on the structures and functions of actin-binding proteins and their roles in live cells. The long-term goal is to understand molecular mechanisms that control the dynamics of the actin cytoskeleton. The work focuses on formin proteins and proteins that coordinate actin filament turnover during endocytosis and cellular motility: Arp2/3 complex, WASp/Scar proteins (activators of Arp2/3 complex), capping protein (terminator of actin filament elongation), ADF/cofilin (recycles actin subunits) and profilin (nucleotide exchange factor for actin and cofactor for formins). Simulations of mathematical models based on quantitative biochemical parameters are compared with quantitative measurements in live cells, not only to impose rigorous thinking but also to identify features that are most likely to distinguish competing hypotheses. The fission yeast, S. pombe, is our experimental organism. Motility of its cortical actin patches depends on the same proteins used by motile amoebas and animal cells to advance their leading edges. The fission yeast offers outstanding genetics, molecular genetics and spectacular cytology, all of which are required to reach the goals of the project. Proteins of interest are manipulated through site directed mutagenesis and studied in live cells after tagging with a fluorescent fusion protein (and verifying their functionality with genetic tests). Methods are available to isolate sufficient quantities of most proteins of interest for structural and biophysical studies. The project has three wide goals for the next five years. The first is to understand how formins change their shape during each cycle that adds a subunit to the end of an actin filament and how force influences the process. The second is to understand how Arp2/3 complex changes its shape from the inactive form free in solution to the active form anchoring an actin filament branch to the side of another actin filament. The third is to use clathrin-mediated endocytosis as the model system to test hypotheses about how actin filaments are assembled and disassembled in live cells. The experiments depend on quantitative fluorescence microscopy to measure dynamics, super-resolution fluorescence microscopy to determine the positions of molecules over time and mathematical modeling to test the ability of biochemical hypotheses to account for events in live cells.